肝癌细胞来源外泌体对肿瘤相关M2型巨噬细胞极化的影响

姚涛; 徐植红; 姚纪友; 夏雨; 陆敏强; 兰天; 刘冰

doi:10.3969/j.issn.1001-5256.2022.03.013

肝癌细胞来源外泌体对肿瘤相关M2型巨噬细胞极化的影响

DOI: 10.3969/j.issn.1001-5256.2022.03.013

姚涛¹,
徐植红¹,
姚纪友²,
夏雨²,
陆敏强²,
兰天¹,
刘冰^1, , ,

1.
广东药科大学药学院，广州 510006
2.
华南理工大学附属第二医院/广州市第一人民医院肝胆二科，广州 510180

基金项目:

药物早期毒性评价创新团队 (2018KCXTD016);

广州市科技计划项目 (202002030387)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：姚涛、徐值红参与课题设计，资料分析，撰写论文；姚纪友、夏雨参与收集数据，修改论文；刘冰、兰天、陆敏强负责课题设计，数据资料分析，指导撰写文章并最后定稿。

详细信息

通信作者:
刘冰，liubing52000@163.com

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出版历程
- 收稿日期: 2021-07-14
- 录用日期: 2021-09-10
- 出版日期: 2022-03-20

Effect of hepatocellular carcinoma cell-derived exosomes on M2 polarization of tumor-associated macrophages

Tao YAO¹,
Zhihong XU¹,
Jiyou YAO²,
Yu XIA²,
Minqiang LU²,
Tian LAN¹,
Bing LIU^{1
, ,
,}

1.
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
2.
Second Department of Hepatobiliary Surgery, The Second Affiliated Hospital of South China University of Technology & Guangzhou First People's Hospital, Guangzhou 510180, China

Research funding:

Innovation Team of Early Drug Toxicity Evaluation (2018KCXTD016);

Guangzhou Science and Technology Program (202002030387)

More Information

Corresponding author: LIU Bing, liubing52000@163.com(ORCID:0000-0002-3144-5073)

摘要

摘要: 目的探讨来源于肝癌细胞的外泌体对肿瘤相关巨噬细胞极化的影响，揭示肝癌形成新机制。方法通过超速离心法分离肝癌细胞来源外泌体，透射电子显微镜、动态光散射粒度分析仪、蛋白免疫印迹法对外泌体表征进行鉴定；诱导巨噬细胞极化模型，实时荧光定量PCR和蛋白免疫印迹法验证其极化状态。符合正态分布的计量资料两组间比较采用t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果透谢电子显微镜显示肝癌细胞来源外泌体为圆形或椭圆形囊泡结构，外泌体粒径大小为(172.65±2.34)nm，蛋白免疫印迹分析显示肝癌细胞来源的外泌体中标志蛋白TSG101和CD63呈高阳性表达。佛波酯15 ng诱导人源单核细胞巨噬细胞24 h贴壁后CD68表达显著增加(6.67±0.98 vs 1.00±0.25，t=11.20，P＜0.001)。蛋白免疫印迹分析显示，相比对照组(L02来源外泌体组)，HCC细胞来源外泌体(低、中、高3种剂量)诱导巨噬细胞表达M2型巨噬细胞标志物Arg-1、CD163均明显增加(P值均＜0.05)。结论肝癌细胞来源外泌体可促进巨噬细胞M2型极化。
- 癌, 肝细胞 /
- 外泌体 /
- 巨噬细胞
Abstract: Objective To investigate the effect of exosomes derived from hepatocellular carcinoma cells on the polarization of tumor-associated macrophages (TAMs), and to reveal the novel mechanism of hepatocellular carcinoma formation. Methods Hepatocellular carcinoma cell-derived exosomes were isolated by ultracentrifugation, and the characteristics of exosomes were identified by transmission electron microscope (TEM), Dynamic Light Scattering (DLS), and Western blotting. The model of macrophage polarization was induced and verified by quantitative real-time PCR and Western blotting. The t-test was used for comparison of normally distributed continuous data between two groups. A one-way analysis of variance was used for comparison between multiple groups, and the LSD-t-test was used for further comparison between two groups. Results TEM showed that hepatocellular carcinoma cell-derived exosomes were round or oval vesicles, LDS showed that the exosomes had a particle size of 172.65±2.34 nm, and Western blotting showed highly positive expression of the biomarkers TSG101 and CD63 in exosomes. There was a significant increase in the expression of CD68 after the addition of 15 ng phorbol ester to induce human-derived mononuclear macrophages for 24 hours to achieve adherent growth (1.00±0.25 vs 6.67±0.98, t=11.20, P < 0.001). Western blotting showed that compared with the control group (L02 cell-derived exosomes), the hepatocellular carcinoma cell-derived exosomes (at low, middle, and high doses) induced M2 polarization of macrophages and increased the expression of the markers Arg-1 and CD163 (all P < 0.05). Conclusion Hepatocellular carcinoma cell-derived exosomes promote M2 polarization of TAMs.
- Carcinoma, Hepatocellular /
- Exosomes /
- Macrophages

HTML全文

图 1 TEM下Hep G2和L02来源外泌体的形态特征

注：a，Hep G2-Exo；b，L02-Exo。

下载: 全尺寸图片幻灯片

图 2 外泌体粒径分布图

下载: 全尺寸图片幻灯片

图 3 蛋白免疫印迹法鉴定外泌体标志分子蛋白表达情况

注：EDS，超速离心分离外泌体后的上层液体。

下载: 全尺寸图片幻灯片

图 4 蛋白免疫印迹法检测M2型巨噬细胞标志蛋白Arg-1、CD163表达

下载: 全尺寸图片幻灯片

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